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Abstract

Introduction

Multiple studies have shown that glial cells of the spinal cord, such as astrocytes
and microglia, have close contact with neurons, suggesting the term tripartite synapse.
In these synapses, astrocytes surrounding neurons contribute to neuronal excitability
and synaptic transmission, thereby increasing nociception and thus the persistence
of chronic pain. Conversely, the N-methyl-D-aspartate (NMDA) receptor is crucial in the generation and maintenance of chronic
pain. It has multiple sites of modulation. One is the site of recognition of extracellular
neurotransmitter (glutamate), which can be blocked by competitive antagonists such
as (3-(2-carboxipiperazin-4)1-propyl phosphonic acid), (±)-CPP, resulting in a blockade
of the calcium current and thus the intracellular transduction process. In the present
study, we investigated whether the potential antinociceptive effect of glial inhibition
produced by propentofylline (PPF) can be enhanced when combined with an NMDA-receptor
inhibitor such as (±)-CPP.

Methods

We used Sprague-Dawley monoarthritic rats. The monoarthritis was induced by injection
of complete Freund adjuvant in the right tibiotarsal joint. Four weeks later, rats
were treated with PPF (1, 10, 30, and 100 μg/10 μl) intrathecally (i.t.) for 10 days,
injected once with (±)-CPP (2.5, 5, 12.5, 25, 50, and 100 μg/10 μl, i.t.), or both
treatments combined. The antinociceptive effect was evaluated on day 11 for PPF and
immediately to (±)-CPP, by assessing the vocalization threshold to mechanical stimulation
of the arthritic paw.

Results

The data indicate that intrathecal administration of increasing concentrations of
(±)-CPP or PPF produced a significant dose-dependent antinociceptive effect with respect
to monoarthritic rats receiving saline. The linear regression analysis showed that
the dose that produces 30% of maximal effect (ED30) for i.t. (±)-CPP was 3.97 μg, and 1.42 μg for i.t. PPF. The administration of the
PPF and (±)-CPP combination in fixed proportions of ED30 produced a dose-dependent antinociceptive effect, showing an interaction of the supraadditive
type.

Conclusions

The results suggest that glia inhibitors can synergically potentiate the effect of
glutamate blockers for the treatment of chronic inflammatory pain.

Introduction

Pain is a sensory modality that, in its acute form, performs the physiological role
of alerting the individual of real or potential tissue damage. It is the immediate
consequence of the pain-pathways activation (nociceptive system), ongoing in a temporal
fashion, and usually resolves when the painful stimulus is removed. Conversely, when
pain lasts, even after the lesion has been healed, or when pain is originated without
apparent tissue damage and lasts for more than 6 months, it is considered pathologic
and called chronic pain [1].

The information collected by the nociceptors is driven by primary afferent fibers
to the spinal cord where they synapse and transmit nociceptive information to projection
neurons located in the dorsal horn of the spinal cord. These projection neurons relay
the information to supraspinal centers through ascending pathways. The first-order
neurons release a number of neurotransmitters, among others, glutamate and substance
P. Substance P stimulates NK-1 receptors that produce a slow and prolonged depolarization
in the projection neuron. Glutamate binds to AMPA receptors, increasing depolarization.
When nociceptive stimulation frequency is greater, it generates a membrane depolarization
that triggers the release of ion Mg2+ from the NMDA receptor [2], promoting the entry of Ca2+ and the subsequent activation of the enzyme nitric oxide synthase, generating nitric
oxide production (NO). NO is a gas that diffuses rapidly through the cell membrane
and acts as an excitatory retrograde messenger in the neurons that generate it, as
in the presynaptic elements and adjacent astrocytes. This event, classified as positive
feedback, has an important role in the development of synaptic neuroplasticity mechanisms,
as has been shown for hippocampal LTP [3] and spinal potentiation known as spinal cord windup, generated against a high and
low frequency of C-fiber stimulation, respectively. As a result, the perception of
pain increases significantly, a potentiation phenomenon in the origin of the generation
of chronic pain.

The NMDA receptors are tetramers [4] that can be assembled in different configurations. The NR1 subunit is essential for
the functionality of the receptor, whereas the NR2 subunits determine the biophysiologic
properties of the channel, like the conductance and the average time of opening or
blocking sensitivity to Mg2+ [5]. The cloning of the receptor subunits revealed that the NR1 subunit has a glycine-binding
site, whereas the NR2 subunit has a glutamate-binding site, which can be blocked by
competitive antagonists such as (±)-CPP, resulting in a blockade of the Ca2+ current, and therefore the intracellular transduction process, as well as the inhibition
of the windup phenomenon [6]. Moreover, a number of other NMDAR antagonists, such as ketamine and ifenprodil acting
on different receptor sites, have been shown to present antinociceptive effects in
models of inflammatory and neuropathic pain [7-11]. This indicates that the (±)-CPP could be used as an analgesic, because this receptor
is involved in the induction and maintenance of central sensitization.

As mentioned earlier, the NMDA receptor is important in the establishment of chronic
pain; however, today we know other factors that can modulate this pain, such as glial
cells [12]. In the last decade, numerous studies have shown that glial cells of the spinal cord
have a close communication with neurons, proposing the term tripartite synapse [13]. This synapse contributes to the modulation of neuronal excitability and synaptic
transmission by increasing nociception and thus the persistence of chronic pain. It
has been found that astrocytes and microglia in the dorsal horn of the spinal cord
are active against a variety of conditions that cause chronic pain and hyperalgesia,
such as subcutaneous swelling, subcutaneous administration of inactivated mycobacterium
[14], and trauma peripheral nerve [15], among others [16].

Once activated glial cells release several neuroactive molecules capable of inducing
or magnifying the pain, such as NO, prostaglandins, arachidonic acid, excitatory amino
acids (glutamate, aspartate, cysteine), quinolinic acid, and growth factors, as well
as variety of proinflammatory cytokines, such as interleukin-1β, interleukin-6, and
tumor necrosis factor [17]. Glial cells and neurons have receptors for cytokines. It is accepted that cytokines
have a role as neuromodulators in the central nervous system, specifically at the
level of second-order nociceptive neurons. In this regard, it has been reported that
IL-1β is able to increase the C-fiber response and windup activity in the spinal cord
[18] at the level of nociceptive afferent terminals, where IL-1β increases the release
of substance P and glutamate [19].

In this context, it is apparent that the main strategy to suppress the communication
between glia and spinal neurons is through the possibility of pharmacologically disrupting
the glial function. In this regard, different drugs have been identified that inhibit
the activity of glia, including propentofylline (PPF) [20]. PPF has inhibitory effects on the activity of phosphodiesterase types I, II, and
IV and on adenosine extracellular transporters in glial cells [21], thereby modifying intracellular cyclic nucleotide homeostasis, leading to a decrease
of the production of proinflammatory cytokines and free radicals in these cells. This
is supported by studies in which has been found an inhibition of the release of tumor
necrosis factor and interleukin 1, as well as the formation of oxygen radicals, in
microglia cultures activated by LPS treatment and subsequently challenged with PPF.
Moreover, increased cAMP-dependent signaling has been shown to increase the expression
of antiinflammatory cytokine IL-10 [22]. Therefore, PPF may increase the production of antiinflammatory cytokines and, in
turn, downregulates the production of proinflammatory cytokines.

PPF also functions as a reuptake inhibitor of adenosine [23]. This is potentially important because adenosine has been proposed to play a role
in neuropathic pain. Adenosine presynaptically inhibits the release of substance P
and glutamate, and postsynaptically decreases the action of substance P and glutamate
[24]. Inhibition of substance P and glutamate release can attenuate central sensitization
and, consequently, could decrease pain.

Because NMDA receptors and glia have an important role in the pathophysiology of chronic
pain, we propose to evaluate whether the coadministration of (±)-CPP and PPF could
enhance the analgesic effect of each drug on chronic inflammatory pain, by using an
isobolographic analysis. The ultimate goal of drug combination is to obtain effective
analgesia with a reduction in the incidence and severity of side effects, which can
be achieved by using lower doses of the drugs [25].

Materials and methods

Animals

In total, 152 male monoarthritic Sprague-Dawley rats (225 to 250 g) were used in this
study. The experimental groups were constituted by six animals in each group. All
animals were obtained from the facilities of the Faculty of Medicine of the University
of Chile, held in a light-dark cycle of 12/12 hours, starting at 8:00 AM, food and
water ad libitum. After each experiment, rats were killed by using an overdose of urethane (3 g/kg,
intraperitoneal, i.p.)

The experiments were conducted in accordance with the "Guide for the Care and Use
of Laboratory Animals of National Institutes of Health (NIH)" [26] and the rules of the International Association for the Study of Pain (IASP) "Models
of animal pain and ethics in experimental animals" [27] and "Ethical standards in research and management of pain." Furthermore, the experimental
protocols were approved by the Bioethics Committee of the Universidad de Santiago
de Chile.

Induction of monoarthritis

Monoarthritic rats were used as a model of chronic inflammatory pain. Monoarthritis
was induced in rats of 120 to 150 g by the method described by Butler et al., [28]. In brief, rats were inoculated with a volume of 50 μl of Freund adjuvant, in the
right ankle joint. The adjuvant consisted of a solution of 60 mg of Mycobacterium butiricum, 6 ml of mineral oil, 4 ml of sodium chloride (0.9%), and 1 ml of Tween 80. Subsequently,
this mixture was autoclaved at 120°C for 20 minutes and stored at room temperature
until use. Before injection, the solution was homogenized by constant stirring. The
injection of adjuvant produces a localized arthritic syndrome that becomes stable
around the fourth week after inoculation, and establishes a persistent pain with hyperalgesia
of the tibiotarsal joint, which is maintained for a period exceeding 2 months. Around
90% to 95% of the injected rats developed mechanical hyperalgesia. Monoarthritic rats
were used between the fourth and the fifth weeks after induction of monoarthritis.

Intrathecal injection

(±)-CPP (Tocris) was administered at single doses of 2.5, 7.5, 12.5, 25, 50, and 100
μg/10 μl. PPF (Sigma) was administered in repeated doses of 1, 10, 30, and 100 μg/10
μl, once daily for a period of 10 days. The two drugs were administered via i.t. injection
in a volume of 10 μl and dissolved in saline; i.t. injection consists of administering
the drug into the subarachnoid space between lumbar vertebrae L5 and L6 [29], by using a Hamilton syringe with a needle 26G × 1/2 inch'. The access to the subarachnoid
space is evidenced by a slight movement in the tail of the rat as a result of the
needle mechanical stimulation penetrating the meninges of the spinal cord. The daily
PPF i.t. injection was done under brief halothane anesthesia (2 minutes).

Experimental groups

To evaluate the antinociceptive effect of both drugs individually on monoarthritic
rats, the vocalization threshold to mechanical stimulation (Randall-Selitto test)
was used. The animals were separated in a first stage of experimentation into two
groups: (a) intrathecal administration of (±)-CPP: 2.5, 7.5, 12.5, 25, 50, or 100
μg/10 μl (n = 6 for each dose); and (b) daily i.t. administration of increasing PPF concentrations
of 1, 10, 30, or 100 μg/10 μl (n = 8 for each dose) for 10 days.

To evaluate the antinociceptive effect of the PPF and (±)-CPP combination, we conducted
a second series of experiments. Both drugs were diluted in decreasing doses (1/3,
1/10, and 1/100) in relation to its ED30. Five groups were used:

1. Daily administration of ED30 of PPF i.t. for 10 days. At day 11, an i.t. injection of ED30 of (±)-CPP was done (n = 6).

2. Daily administration of ED30 of PPF i.t. for 10 days. At day 11, an i.t. injection of 1/3 of ED30 of (±)-CPP was done (n = 6).

3. Daily administration of ED30 of PPF i.t. for 10 days. At day 11, an i.t. injection of 1/10 of ED30 of (±)-CPP was done (n = 6).

4. Daily administration of ED30 of PPF i.t. for 10 days. At day 11, an i.t. injection of 1/30 of ED30 of (±)-CPP was done (n = 6).

5. Daily administration of ED30 of PPF i.t. for 10 days. At day 11, an i.t. injection of 1/100 of the ED30 of (±)-CPP was done (n = 6).

Controls were provided by normal and monoarthritic rats receiving saline, as follows:

1. Normal group of the same age of monoarthritic rats, receiving i.t. injection of
saline before testing (n = 6).

2. Monoarthritic saline group, pooled from saline controls for the (±)-CPP, PPF, and
combined (±)-CPP/PPF series, receiving i.t. daily injection of saline for a period
of 10 days, followed by an i.t. injection of saline at day 11, or a single injection
at day 11 (n = 16). The three groups were pooled because they showed no significant differences
in vocalization threshold between them at any time of testing.

Mechanical hyperalgesia

This behavioral test consists of adding a continuous and increasing pressure with
a taper ending in blunt tip on the posterior knee joint of the rat to generate a nociceptive
behavior. The response is evidenced by a vocalization or withdrawal reflex of the
limb in response to stimulation. The pressure on the joint is increased gradually
(linearly) up to 570 g, a value that does not harm the animal. The equipment used
for this test was called analgesiometer Ugo Basile. Each animal was tested 2 times
at 5, 15, 30, and 60 min for monoarthritic rats treated with (±)-CPP or the combination
of PPF and (±)-CPP, and at 15, 30, and 60 min for monoarthritic PPF-treated rats.
After the experiment, all rats were killed with an overdose of urethane. Grams of
pressure, which expresses rat nociceptive behavior, were saved for later analysis.
The data were expressed as percentage change to baseline and were then averaged over
the different groups and different times. Later, the area under the curve (AUC) was
calculated, by using the Microcal Origin V 6.0 program, and the groups were compared
statistically.

Isobolographic analysis

The evaluation of the interaction between both drugs was performed by using isobolographic
analysis [25]. The isobologram is a graphic method that consists of calculating the theoretic additive
dose for each level of effect and their statistical comparison with the combination
dose that produces the same effect experimentally. Equieffective doses of both drugs
alone are needed to calculate the expected dose in a combination. To this end, we
determined the dose that produces 30% of maximal effect (ED30) by using a linear regression analysis from the dose-response curve of six increasing
doses of (±)-CPP and the previously mentioned for increasing doses of PPF. Once we
obtained the ED30 of both drugs, a graph was constructed by placing in the y-axis of the ED30 point of (±)-CPP and the x-axis point of the ED30 of PPF. The union of two points by a straight line (isobolo), also known as a line
of additivity or no interaction, helped to establish the type of interaction (synergism
or antagonism) of both compounds. The interaction between both drugs was carried out
by an administration of 1, 1/3, 1/10, 1/30, and 1/100 of the ED30 (±)-CPP, and PPF. The coadministration was performed through intrathecal PPF ED30 daily for 10 days. The antinociception was assessed on day 11 with the Randall-Selitto
test and then followed by i.t. administration of ED30 (±)-CPP; antinociception was assessed by the same test. Then the ED30 of the association of both drugs (ED30 experimental), from a dose-response curve, was obtained by linear regression analysis.
This dose was compared statistically with the dose that theoretically represents the
simple addition of effects, obtained by the following formula:

Where R is the power ratio between the two drugs given alone, P1 is the proportion
of the drug (PPF) in the mixture, and P2 is the proportion of drug 2 ((±)-CPP) in
the mixture.

The graphic region in which is located the experimental value (ED30 experimental) in relation to the theoretic value (ED30 theoretic additivity) determines the type of interaction: If the value is located
under the line of additivity and is statistically different from the theoretic value,
the type of interaction is synergistic or supraadditive (effect greater than the sum
of the individual effects of drugs); if located next to the line of additivity and
not statistically different from the theoretic value, the interaction is simple additivity
(equal effect of the sum of each drug); conversely, if the experimental value lies
above the line of additivity and is statistically different from the theoretic nature
of the interaction, it is subadditive or antagonistic. At the same time, we calculated
the interaction index (I.I.) between the drugs, obtained from the following formula:

This index, when less than 1 corresponds to a synergistic interaction, when equal
to 1, corresponds to an additive interaction, and when greater than 1 is an antagonistic
interaction [30].

Statistical analysis

The results were expressed as mean percentage of antinociceptive effect ± standard
error of the mean (SEM) for each experimental group, from baseline obtained before
the injection of saline or each of the drugs under study, as appropriate. The quantification
of the antinociceptive effect (%AE) of the drugs tested were calculated as a percentage
change in AUC from baseline (basal) for each rat, and set a maximum pressure cut-off
of 570 g in the Randall-Selitto, according to the following formula:

(1)

(2)

Where AUCpre and AUCpost are approximate integrals of the curves obtained by the method of trapezoids and pre-post
drug injection, respectively, according to Eq. 1. The AUCdrug effect values are the integrals of the real effect of the drug. The antinociceptive effect
(AE) was calculated according to Eq. 2, where the AUCcut-off corresponds to the area of maximum pressure possible on the animal.

To analyze the time-course of the antinociceptive effect of increasing doses of i.t.
(±)-CPP and PPF, two-way ANOVA was performed. It allowed us to assess both intergroup
comparisons (vocalization-threshold changes under different treatments) and intragroup
comparisons (vocalization thresholds along the time), followed by the Bonferroni multiple
comparisons test. To analyze the percentage antinociception obtained from the area
under the time-course curves, one-way ANOVA was used, followed by Tukey-Kramer multiple
comparisons test. To assess differences for the theoretic ED30 and experimental ED30, the two-tailed Student t test was used. All statistical analyses were performed with the Prism 3.0 software
(GraphPad Software, Inc., San Diego CA, USA).

Results

Dose-response of (±)-CPP on mechanical nociception in monoarthritic rats

The administration of (±)-CPP (2.5, 5, 12.5, 25, 50, or 100 μg/10 μl) increased the
vocalization threshold measured at 5, 15, 30, and 60 min after injection compared
with rats receiving saline (Figure 1A), well above the pre-monoarthritis threshold. Areas under curves indicate that rats
administered with saline showed a percentage of antinociception of 1.1% ± 1.4%, whereas
rats administered with increasing doses of (±)-CPP showed a percentage of antinociception
of 26.0% ± 2.4%, 33.9% ± 4.5%, 43.2% ± 5.0%, 47.8% ± 5.2%, 54.4% ± 6.8%, and 67.0%
± 6.8%, respectively (Figure 1B). In all cases, they were significantly higher than the percentages represented by
the saline, showing a dose-dependent increase in trend. The linear regression analysis
of the percentage AE showed that the ED30 was 3.97 μg, with a 95% confidence interval (95% CI) of 2.35 to 6.7 μg.

Figure 1.Antinociceptive effect of (±)-CPP in monoarthritic rats. (A) Time-course of the antinociceptive effect of increasing doses of i.t. (±)-CPP (2.5,
5.0, 12.5, 25, 50, and 100 μg/rat). Vocalization thresholds were measured before (left
arrow), and then 28 days after monoarthritis induction, and after a single injection
of CPP. Open symbols, values from monoarthritic rats. Solid symbols, values from normal
rats receiving saline under a similar protocol. The right arrow corresponds to CPP
or saline injection. Values are expressed as mean ± standard error of the mean (SEM);
n = 6 rats per group. Two-way ANOVA indicates a significant effect for the (±)-CPP Treatment
factor (F(6, 175) = 39.32; ANOVA P < 0.0001), as well as for the Time factor (F(5, 175) = 56.64; ANOVA P < 0.0001). Bonferroni multiple comparisons post hoc test showed that vocalization
thresholds of all (±)-CPP treated rats (2.5, 5.0, 12.5, 25, 50, and 100 μg/rat) were
significantly higher (p < 0.05) than the corresponding threshold of saline-treated
animals (symbols omitted). In addition, Bonferroni multiple comparisons post hoc test showed that vocalization thresholds of rats after receiving the four highest
doses of (±)-CPP were significantly higher (*P < 0.05) than the threshold measured before monoarthritis induction. (B) Ordinate indicates percentage antinociception obtained from the area under the time-course
curves from (A) (see Materials and methods). Data are expressed as mean ± standard
error of the mean (SEM), and were analyzed by using one-way ANOVA followed by Tukey-Kramer
multiple comparisons test (*P < 0.05; **P < 0.01; ***P < 0.001; compared with monoarthritic rats receiving saline).

Dose-response of PPF on mechanical nociception in monoarthritic rats

Unlike the study with (±)-CPP, the PPF was administered over a longer term (that is,
once daily for 10 consecutive days) to ensure that the glia became inactive. At day
11 of saline or PPF treatment, the animals were challenged with a single dose of saline
(10 μl) and studied at 0, 15, 30, and 60 minutes after injection. The effect of PPF
was evaluated by comparing the treatments as independent groups.

The administration of saline i.t. for 10 days in monoarthritic rats produced an average
threshold of vocalization at zero time of 174 ± 9.2 g. After the injection of saline
challenge, this vocalization threshold was unchanged at 0, 15, 30, and 60 minutes
after injection (Figure 2A). In the groups treated with 1, 10, 30, and 100 μg/10 μl PPF for 10 days, the vocalization
threshold at 0 time was 183 ± 6.3, 226 ± 13.5, 288 ± 10.0, and 310 ± 8.8 g, respectively,
which remained without modifications during the 60 minutes of measurement. These data
show that PPF produced dose-dependent increases in the vocalization threshold in monoarthritic
rats, the two higher doses raising the threshold above those observed in the premonoarthritis
condition.

Figure 2.Antinociceptive effect of PPF in monoarthritic rats. (A) Time course of the antinociceptive effect of daily repeated injections of PPF in monoarthritic rats. Vocalization thresholds were measured before (left arrow),
and then 28 days after monoarthritis induction, and 10 days after repeated injection
of PPF (right arrows) (Time 0). Empty symbols represent values from monoarthritic
rats. Solid symbols represent values from normal rats receiving saline under a similar
protocol. Values are expressed as mean ± standard error of the mean (SEM); n = 6 rats per group. Two-way ANOVA indicates a significant effect for the PPF treatment
factor (F(4, 125) = 132.20; ANOVA P < 0.0001) as well as for the time factor (F(4, 125) = 7.55; ANOVA P < 0.0001). Bonferroni multiple comparisons post hoc test showed that vocalization thresholds of rats receiving the three highest doses
of PPF (10, 30, and 100 μg/rat) were significantly higher (P < 0.05) than the corresponding thresholds of saline-treated animals (symbols omitted).
In addition, Bonferroni multiple comparisons post hoc test showed that vocalization thresholds of rats after receiving the highest doses
of PPF were significantly higher (*P < 0.05) than the threshold measured before monoarthritis induction. (B) Ordinate indicates percentage antinociception obtained from the area under the time-course
curves from (A) (see Materials and methods). Data are expressed as mean ± standard
error of the mean (SEM), and were analyzed by using one-way ANOVA followed by Tukey-Kramer
multiple comparisons test (***P < 0.001, compared with monoarthritic rats receiving saline).

Area under curves indicates that monoarthritic rats injected with increasing doses
of PPF (1, 10, 30, or 100 μg/10 μl) showed a percentage of antinociception of 32.8%
± 1.0%, 39.4% ± 2.4%, 50.1% ± 1.8%, and 54.4% ± 1.6%, respectively (Figure 2B), which were significantly higher than that observed in saline controls. Linear regression
analysis allowed calculation of an ED30 of 1.42 μg with a 95% CI of 0.88 to 2.27 μg.

Dose-response of the combination of PPF and (±)-CPP: isobolographic study

In a second series of experiments, (±)-CPP and PPF were administered together in a
proportion obtained from their respective ED30, which made possible to calculate the theoretic additive dose, generating a series
of theoretic doses shown in Table 1.

Table 1. Fixed proportions, equieffective and theoretically additive, used for the combination
of both drugs

In the five groups treated with increasing doses of the PPF/(±)-CPP combination (according
to Table 1), the vocalization threshold increased for all doses, starting at 5 minutes, and
remained elevated until 60 minutes after injection (Figure 3A). For the three higher doses of the combination, the vocalization threshold remained
above the premonoarthritis threshold throughout the testing period.

Figure 3.Antinociceptive effect of PPF/(±)-CPP combination in monoarthritic rats. (A) Time course of the antinociceptive effect of daily repeated injections of PFF followed
by a single injection of (±)-CPP in monoarthritic rats. Vocalization thresholds were
measured before (left arrow) and then 28 days after monoarthritis induction, and after
10 repeated and a single injection of PPF and (±)-CPP, respectively (right arrows)
(Time 0). Open symbols represent values from monoarthritic rats. Solid symbols represent
values from normal rats receiving saline under a similar protocol. Values are expressed
as mean ± standard error of the mean (SEM); n = 6 rats per group. Two-way ANOVA indicates a significant effect for the PPF/(±)-CPP
treatment factor (F(5, 180) = 51,78; ANOVA P < 0.0001) as well as for the time factor (F(5, 180) = 44.37; ANOVA P < 0.0001). Bonferroni multiple comparisons post hoc test showed that vocalization thresholds of all PPF/(±)-CPP treated rats were significantly
higher (P < 0.05) than the corresponding threshold of saline-treated animals (symbols omitted).
In addition, Bonferroni multiple-comparisons post hoc test showed that vocalization thresholds of rats after receiving the highest doses
of PPF/(±)-CPP were significantly higher (*P < 0.05) than the threshold measured before monoarthritis induction. (B) Ordinate indicates percentage antinociception obtained from the area under the time-course
curves from (A) (see Materials and methods). Data are expressed as mean ± standard
error of the mean (SEM), and were analyzed by using one-way ANOVA followed by Tukey-Kramer
multiple comparisons test (**P < 0.01; ***P < 0.001; compared with monoarthritic rats receiving saline).

The %AE (Figure 3B) indicates that monoarthritic rats injected with equieffective doses of the PPF/(±)-CPP
combination showed a percentage of antinociception of 29.1% ± 5.0%, 32.1% ± 1.9%,
40.8% ± 7.9%, 36.9% ± 7.4%, and 45.0% ± 3.6%, which were significantly higher than
that observed in saline controls. Linear regression analysis showed that the ED30 for the PPF/(±)-CPP combination was 0.063 μg with a 95% CI of 0.012 to 0.334 μg.

The combined effect of both drugs was analyzed by constructing an isobologram graph
(Figure 4), which shows that the antinociceptive activity induced by coadministration of fixed
proportions of the ED30 for PPF and (±)-CPP produced a greater antinociceptive effect than a simple additivity
in monoarthritic rats. This result is achieved because the ED30 point for the combination is under the curve of isobolo and statistically different
from the ED30 theoretically additive, indicating that the effect of the combination of both drugs
is supraadditive (t = 2.879; P < 0.001, two-tailed Student t test). The interaction index between PPF and (±)-CPP was 0.024.

Figure 4.Isobologram for the coadministration of PPF and (±)-CPP at fixed-ratio combinations. The white circle on the straight line represents the point of theoretic activity
calculated with a confidence limit of 95%, whereas the black circle under the straight
line corresponds to the experimental point obtained with the monoarthritic rats (95%
confidence limit). The experimental point was significantly different from the theoretically
calculated point (mean ± SEM; ***P < 0.001, two-tailed Student t test), indicating supraadditive synergy in the Randall-Selitto test.

Discussion

The results of this study show that the analgesic effect observed by combining PPF
(a glial cells inhibitor) and (±)-CPP (an NMDA-receptor antagonist) on the paw-pressure
test is supraadditive, in rats with chronic inflammatory pain. The ED30 obtained for (±)-CPP was 3.97 μg, and for PPF, 1.42 μg, whereas the ED30 of the combination was 0.063 μg, which was significantly lower than that expected
by simple additivity. The ED50 was not used because the maximum effect of the drugs administered separately did not
exceed 60% of the maximum effect.

As pointed out elsewhere [31-34], a supraadditive effect of combining two drugs producing the same effect could occur
only if the mechanisms of action involved are totally or partially different (that
is, "purely mutually nonexclusive" or "partially or nonpurely nonexclusive," as defined
by Chou [31]), but not when the mechanism of action is the same for the two combined drugs. In
the case of combining PPF and (±)-CPP, the mechanisms of action are partially independent
and therefore consistent with the supraadditive effect found in the present study.

Some evidence supports that the administration of PPF to cultures of microglia from
neonatal rat brain, activated by lipopolysaccharides, inhibits secretion of tumor
necrosis factor (TNF-α), interleukin 1 (IL-1), and oxygen radicals [21]. Similar results obtained from microdialysis in the lumbar spinal cord of rats submitted
to sciatic nerve chronic constriction injury have been reported [35]. It seems that inhibition by PPF of glial proinflammatory cytokine secretion is mediated
by the cAMP-PKA pathway, because PPF effects are mimicked by dibutyryl-cAMP [36], and cAMP-PKA signaling represses proinflammatory cytokine gene expression in microglia
[37]. However, the mechanisms of action of PPF are not yet clear. For instance, PPF has
been shown to reinstate the decreased expression of glutamate transporters GLT-1 and
GLAST produced for the L5 nerve transection in mice [38], thus promoting glial glutamate uptake and thereby glutamate excitotoxicity, therefore
decreasing nociception by a mechanism different from proinflammatory cytokine repression.
Furthermore, it has been reported that PPF decreases hyperalgesia induced by intracisternal
BDNF administration [39], which may constitute another different mechanism from the previously mentioned.
BDNF synthesis is increased not only in primary afferents during chronic pain [40,41] but also in second-order nociceptive neurons [42,43] and glial cells [44,45] of the dorsal horn. It has been claimed that BDNF promotes pain through two different
mechanisms: (a) by potentiating the glutamatergic transmission in the spinal cord
via increased glutamate release and enhanced synaptic efficacy at the postsynaptic
level [46], and (b) by reducing the expression of the KCC2 transporter in dorsal horn neurons,
which leads to a shift in the transmembrane anion gradient that causes normally inhibitory
anionic synaptic currents to be excitatory; this latter mechanisms has been reported
to be triggered only by glial-derived BDNF neurotrophin [44]. Because expression of the KCC2 transporter was found to be significantly reduced
in spinal cord slices of rats with chronic inflammatory pain [47], it is likely that in the present study, PPF could reduce hyperalgesia by depressing
glial BDNF release, thereby restoring the normal transmembrane anion gradient.

Conversely, it is accepted that the NMDA receptor is crucial in the transfer of nociceptive
information in the spinal cord, specifically between the first and second nociceptive
projection neurons [48]. Studies using antagonists of NMDA receptors have demonstrated their effectiveness
as antinociceptive drugs in animal models of central hypersensitivity induced by cutaneous
application of the chemical irritant mustard oil, tested with brief electrical stimulation
of the sural nerve and challenged with MK-801 and (±)-CPP [49]. For example, MK-801 (an uncompetitive antagonist of the NMDA receptor) prevents
skin and tactile hyperalgesia induced by muscle noxious C-fiber stimuli [7,50], and (±)-CPP (a competitive antagonist of the glutamate-binding site on the NMDA
receptor) specifically blocks the action of glutamate, thus producing analgesia in
different pain models [12,51]. In the present study, we demonstrated that increasing doses of (±)-CPP have a dose-dependent
antinociceptive effects in monoarthritic rats.

Thus, it seems clear that PPF and (±)-CPP act through different mechanisms, but it
is also clear that PPF and (±)-CPP can functionally interact because PPF lowers glial
release of BDNF, thus avoiding the potentiating effect of the glial-derived BDNF on
the glutamatergic transmission in the spinal cord. Therefore, the antihyperalgesic
mechanisms of action of PPF and (±)-CPP are only partially independent, because PPF-
and (±)-CPP-dependent effects can converge at the NMDA-receptor functionality, thus
supporting supraadditive interactions when combined in equieffective doses.

Conclusions

We showed for the first time that the glial inhibitor PPF can synergistically potentiate
the effect of (±)-CPP, a drug that inhibits NMDA-receptor activity, thus opening the
field of associating glial inhibitors to NMDA-receptor blockers in the pharmacologic
treatment of chronic inflammatory pain. Glial inhibitors [52,53] and NMDA antagonists [54,55] have been associated with opioid therapy in a variety of painful conditions, but
glial inhibitors and NMDA antagonists have not still assayed in combination clinical
studies.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

FM, TP, and CL performed most of the experiments. TP performed experiments in inducing
monoarthritis. LC, TP, AH, and CL conceived the study and participated in the design,
in the interpretation of results, and in drafting the manuscript. All authors read
and approved the final manuscript.

Acknowledgements

This study was supported by grants DICYT 011043LF, FONDECYT 1070115, and CEDENNA FB0807.